Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39.

A precise understanding of the genomic organization into transcriptional units and their regulation is essential for our comprehension of opportunistic human pathogens and how they cause disease. Using single-molecule real-time (PacBio) sequencing we unambiguously determined the genome sequence of Streptococcus pneumoniae strain D39 and revealed several inversions previously undetected by short-read sequencing. Significantly, a chromosomal inversion results in antigenic variation of PhtD, an important surface-exposed virulence factor. We generated a new genome annotation using automated tools, followed by manual curation, reflecting the current knowledge in the field. By combining sequence-driven terminator prediction, deep paired-end transcriptome sequencing and enrichment of primary transcripts by Cappable-Seq, we mapped 1015 transcriptional start sites and 748 termination sites. We show that the pneumococcal transcriptional landscape is complex and includes many secondary, antisense and internal promoters. Using this new genomic map, we identified several new small RNAs (sRNAs), RNA switches (including sixteen previously misidentified as sRNAs), and antisense RNAs. In total, we annotated 89 new protein-encoding genes, 34 sRNAs and 165 pseudogenes, bringing the S. pneumoniae D39 repertoire to 2146 genetic elements. We report operon structures and observed that 9% of operons are leaderless. The genome data are accessible in an online resource called PneumoBrowse (https://veeninglab.com/pneumobrowse) providing one of the most complete inventories of a bacterial genome to date. PneumoBrowse will accelerate pneumococcal research and the development of new prevention and treatment strategies.

High-resolution analysis of the pneumococcal transcriptome under a wide range of infection-relevant conditions.

Streptococcus pneumoniae is an opportunistic human pathogen that typically colonizes the nasopharyngeal passage and causes lethal disease in other host niches, such as the lung or the meninges. The expression and regulation of pneumococcal genes at different life-cycle stages, such as commensal or pathogenic, are not entirely understood. To chart the transcriptional responses of S. pneumoniae, we used RNA-seq to quantify the relative abundance of the transcriptome under 22 different infection-relevant conditions. The data demonstrated a high level of dynamic expression and, strikingly, all annotated pneumococcal genomic features were expressed in at least one of the studied conditions. By computing the correlation values of every pair of genes across all studied conditions, we created a co-expression matrix that provides valuable information on both operon structure and regulatory processes. The co-expression data are highly consistent with well-characterized operons and regulons, such as the PyrR, ComE and ComX regulons, and have allowed us to identify a new member of the competence regulon. Lastly, we created an interactive data center named PneumoExpress (https://veeninglab.com/pneumoexpress) that enables users to access the expression data as well as the co-expression matrix in an intuitive and efficient manner, providing a valuable resource to the pneumococcal research community.

Refining the Pneumococcal Competence Regulon by RNA Sequencing.

Competence for genetic transformation allows the opportunistic human pathogen Streptococcus pneumoniae to take up exogenous DNA for incorporation into its own genome. This ability may account for the extraordinary genomic plasticity of this bacterium, leading to antigenic variation, vaccine escape, and the spread of antibiotic resistance. The competence system has been thoroughly studied, and its regulation is well understood. Additionally, over the last decade, several stress factors have been shown to trigger the competent state, leading to the activation of several stress response regulons. The arrival of next-generation sequencing techniques allowed us to update the competence regulon, the latest report on which still depended on DNA microarray technology. Enabled by the availability of an up-to-date genome annotation, including transcript boundaries, we assayed time-dependent expression of all annotated features in response to competence induction, were able to identify the affected promoters, and produced a more complete overview of the various regulons activated during the competence state. We show that 4% of all annotated genes are under direct control of competence regulators ComE and ComX, while the expression of a total of up to 17% of all genes is affected, either directly or indirectly. Among the affected genes are various small RNAs with an as-yet-unknown function. Besides the ComE and ComX regulons, we were also able to refine the CiaR, VraR (LiaR), and BlpR regulons, underlining the strength of combining transcriptome sequencing (RNA-seq) with a well-annotated genome.IMPORTANCEStreptococcus pneumoniae is an opportunistic human pathogen responsible for over a million deaths every year. Although both vaccination programs and antibiotic therapies have been effective in prevention and treatment of pneumococcal infections, respectively, the sustainability of these solutions is uncertain. The pneumococcal genome is highly flexible, leading to vaccine escape and antibiotic resistance. This flexibility is predominantly facilitated by competence, a state allowing the cell to take up and integrate exogenous DNA. Thus, it is essential to obtain a detailed overview of gene expression during competence. This is stressed by the fact that administration of several classes of antibiotics can lead to competence. Previous studies on the competence regulon were performed with microarray technology and were limited to an incomplete set of known genes. Using RNA sequencing combined with an up-to-date genome annotation, we provide an updated overview of competence-regulated genes.

Bright fluorescent Streptococcus pneumoniae for live-cell imaging of host-pathogen interactions.

Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis.

Klebsiella pneumoniae OmpR facilitates lung infection through transcriptional regulation of key virulence factors.

IMPORTANCE: Bacteria use two-component regulatory systems (TCSs) to adapt to changes in their environment by changing their gene expression. In this study, we show that the EnvZ/OmpR TCS of the clinically relevant opportunistic pathogen Klebsiella pneumoniae plays an important role in successfully establishing lung infection and virulence. In addition, we elucidate the K. pneumoniae OmpR regulon within the host. This work suggests that K. pneumoniae OmpR might be a promising target for innovative anti-infectives.

Pneumolysin promotes host cell necroptosis and bacterial competence during pneumococcal meningitis as shown by whole-animal dual RNA-seq.

Pneumolysin is a major virulence factor of Streptococcus pneumoniae that plays a key role in interaction with the host during invasive disease. How pneumolysin influences these dynamics between host and pathogen interaction during early phase of central nervous system infection in pneumococcal meningitis remains unclear. Using a whole-animal in vivo dual RNA sequencing (RNA-seq) approach, we identify pneumolysin-specific transcriptional responses in both S. pneumoniae and zebrafish (Danio rerio) during early pneumococcal meningitis. By functional enrichment analysis, we identify host pathways known to be activated by pneumolysin and discover the importance of necroptosis for host survival. Inhibition of this pathway using the drug GSK'872 increases host mortality during pneumococcal meningitis. On the pathogen's side, we show that pneumolysin-dependent competence activation is crucial for intra-host replication and virulence. Altogether, this study provides new insights into pneumolysin-specific transcriptional responses and identifies key pathways involved in pneumococcal meningitis.

In vivo dual RNA-seq reveals that neutrophil recruitment underlies differential tissue tropism of Streptococcus pneumoniae.

Streptococcus pneumoniae is a genetically diverse human-adapted pathogen commonly carried asymptomatically in the nasopharynx. We have recently shown that a single nucleotide polymorphism (SNP) in the raffinose pathway regulatory gene rafR accounts for a difference in the capacity of clonally-related strains to cause localised versus systemic infection. Using dual RNA-seq, we show that this SNP affects expression of bacterial genes encoding multiple sugar transporters, and fine-tunes carbohydrate metabolism, along with extensive rewiring of host transcriptional responses to infection, particularly expression of genes encoding cytokine and chemokine ligands and receptors. The data predict a crucial role for differential neutrophil recruitment (confirmed by in vivo neutrophil depletion and IL-17 neutralization) indicating that early detection of bacteria by the host in the lung environment is crucial for effective clearance. Thus, dual RNA-seq provides a powerful tool for understanding complex host-pathogen interactions and reveals how a single bacterial SNP can drive differential disease outcomes.

Time-resolved dual RNA-seq reveals extensive rewiring of lung epithelial and pneumococcal transcriptomes during early infection.

BACKGROUND: Streptococcus pneumoniae, the pneumococcus, is the main etiological agent of pneumonia. Pneumococcal infection is initiated by bacterial adherence to lung epithelial cells. The exact transcriptional changes occurring in both host and microbe during infection are unknown. Here, we developed a time-resolved infection model of human lung alveolar epithelial cells by S. pneumoniae and assess the resulting transcriptome changes in both organisms simultaneously by using dual RNA-seq. RESULTS: Functional analysis of the time-resolved dual RNA-seq data identifies several features of pneumococcal infection. For instance, we show that the glutathione-dependent reactive oxygen detoxification pathway in epithelial cells is activated by reactive oxygen species produced by S. pneumoniae. Addition of the antioxidant resveratrol during infection abates this response. At the same time, pneumococci activate the competence regulon during co-incubation with lung epithelial cells. By comparing transcriptional changes between wild-type encapsulated and mutant unencapsulated pneumococci, we demonstrate that adherent pneumococci, but not free-floating bacteria, repress innate immune responses in epithelial cells including expression of the chemokine IL-8 and the production of antimicrobial peptides. We also show that pneumococci activate several sugar transporters in response to adherence to epithelial cells and demonstrate that this activation depends on host-derived mucins. CONCLUSIONS: We provide a dual-transcriptomics overview of early pneumococcal infection in a time-resolved manner, providing new insights into host-microbe interactions. To allow easy access to the data by the community, a web-based platform was developed ( http://dualrnaseq.molgenrug.nl ). Further database exploration may expand our understanding of epithelial-pneumococcal interaction, leading to novel antimicrobial strategies.